Ion channels regulate a diversity of cellular functions through generation of ionic currents, including cardiac, CNS, and immune physiology. It is estimated that between 5-30% of marketed drugs may regulate ion channel activity (Overington et al., Nat Reviews Drug Discovery 5:993-6, 2006). Subfamily selectivity is a desired feature of new therapeutics to improve efficacy and safety of current non-selective drugs, and poses a significant challenge for small molecules and known naturally occurring peptide toxins (Wickenden et al., Future Med Chem 4:661-79, 2012). This is especially true within large homologous families such as voltage-gated K+, Ca+ and Na+ channels.
Kv1.3, the potassium voltage-gated channel subfamily A member 3, is expressed on T cells and functions to regulate T cell activation. Sustained calcium signaling is required for T cell activation for upregulation of cell surface activation markers and increase in cytokine production and proliferation via calcineurin dependent dephosphorylation and nuclear translocation of nuclear factor of activated T cells (NFAT). Inositol triphosphate (IP3) dependent release of internal calcium stores from the endoplasmic reticulum activates the calcium release activated calcium channels (CRAC) on the cell surface, providing an influx of extracellular calcium and sustained calcium signaling (reviewed in Cahalan et al., Immunol Rev 231:59-87, 2009). An efflux of potassium is required for the cells to remain in a hyperpolarized state and for calcium influx to be maintained for full T cell activation. This potassium efflux appears to be regulated through the voltage-gated potassium channel Kv1.3 and the calcium-activated potassium channel KCa3.1. Blockers selective for Kv1.3 have demonstrated that Kv1.3 is the potassium channel responsible for regulating calcium signaling, even in the absence of any inhibition of KCa3.1. (Beeton et al., Mol Pharmacol 67:1369-81, 2005). Blocking Kv1.3 depolarizes T cells and inhibits calcium entry, cytokine production, and proliferation of activated T cells in vitro (reviewed in Cahalan et al., Immunol Rev 231:59-87, 2009).
Kv1.3 blockers have been shown to reduce T cell dependent disease progression in autoimmune models, such as experimental autoimmune encephalomyelitis (EAE), experimental arthritis, delayed-type hypersensitivity (DTH), allergic contact dermatitis and glomerulonephritis (Rangaraju et al., Expert Opin Ther Targets 13:909-24, 2009; Beeton et al., Proc Natl Acad Sci USA. 103:17414-9, 2006; Koo et al., J Immunol 158:5120-8, 1997; Hyodo et al., Am J Physiol Renal Physiol 299:F1258-69, 2010). The calcium calcineurin NFAT pathway inhibitors cyclosporine A (Neoral, Sandimmune, Gengraf) and Tacrolimus (FK-506 or fujimycin) are approved treatments for severe immune disorders, including transplant rejection and severe rheumatoid arthritis. The broad distribution of calcineurin in tissues such as kidneys may result in a higher degree of mechanism based toxicity, narrow safety margins, and limited therapeutic application for these compounds. T cell inhibition using selective Kv1.3 blockers may result in increased safety profile and greater efficacy in the treatment of T cell mediated inflammatory and autoimmune diseases.
Kv1.3 may play a role in regulating weight gain and improving insulin sensitivity. Kv1.3 deficient mice show reduced weight gain, higher insulin sensitivity, and reduced plasma glucose levels (Xu et al., Hum Mol Genet 12:551-9, 2003). Kv1.3 blockers have been shown to increase glucose transporter 4 (GLUT4) cell surface expression in skeletal muscle and adipose tissue, and result in increased insulin sensitivity in normal and ob/ob obese mice, and to increase glucose uptake in primary adipocytes in vitro (Xu et al., Proc Natl Acad Sci USA 101:3112-7, 2004). In humans, a single nucleotide polymorphism (SNP) in the Kv1.3 gene has been associated with decreased insulin sensitivity and impaired glucose tolerance (Tschritter, Clin Endocrinol Metab 91:654-8, 2006).
Kv1.3 may have a critical function in smooth muscle proliferative disorders like restenosis in patients following vascular surgery, such as angioplasty. Kv1.3 expression is increased in proliferating human and mouse smooth muscle cells. Kv1.3 blockers inhibit calcium entry, reduce smooth muscle cell migration, and inhibit neointimal hyperplasia in ex vivo human vein samples (Cheong et al., Cardiovasc Res 89:282-9, 2011).
Increasing evidence indicates that Kv1.3 channels are involved in the activation and/or proliferation of many types of cells, including tumor cells (Bielanska et al., Curr Cancer Drug Targets 9:904-14, 2009), microglia (Khanna et al., Am J Physiol Cell Physiol 280:C796-806, 2001) and differentiation of neuronal progenitor cells (Wang et al., J Neurosci 30:5020-7, 2010) suggesting that Kv1.3 blockers may be beneficial in the treatment of neuroinflammatory and neurodegenerative diseases, and cancers.
Toxin peptides produced by a variety of organisms have evolved to target ion channels. Snakes, scorpions, spiders, bees, snails, sea anemone, insects, arachnids, cnidarians, reptiles, and mollusks are a few examples of organisms that produce venom that can serve as a rich source of small bioactive toxin peptides or “toxins” that potently and selectively target ion channels and receptors. In most cases, these toxin peptides have evolved as potent antagonists or inhibitors of ion channels, by binding to the channel pore and physically blocking the ion conduction pathway or by antagonizing channel function by binding to a region outside the pore (e.g., the voltage sensor domain). Toxins peptides are typically about 20-80 amino acids long with distinct disulfide bond pairing, and can be divided into a number of superfamilies based on their disulfide connections and peptide folds. Many venom toxins are being engineered to improve their properties such as selectivity (King, Expert Opin Biol Ther 11:1469-84, 2011; Escoubas and King, Expert Review Proteomics 6:221-4, 2009).
Venom peptides demonstrating Kv1.3 blocking include ShK, OdK2, OsK1, margatoxin, kaliotoxin etc (see Chandy et al., Trends in Pharmacol Sci 25:280-9, 2004). Kv1.3 blockers OdK2 and OsK1 (alpha-KTx3.7) are homologous members of the α-KTx3 scorpion toxin family from the venom of Odontobuthus doriae and Orthochirus scrobiculosus, respectively (Abdel-Mottaleb et al., Toxicon 51:1424-30, 2008; Mouhat et al., Biochem J 385(Pt 1):95-104, 2005; Int. Pat. Publ. No. WO2006/002850). OsK1 (alpha-KTx3.7) was reported to block Kv1.3, Kv1.1 and Kv1.2 channels potently and KCa3.1 channel moderately (Mouhat et al., Biochem J 385(Pt 1):95-104, 2005). OdK2 (alpha-KTx3.11) was reported to block Kv1.3 while having no activity on Kv1.1, Kv1.2, Kv1.4, Kv1.5, and Kv1.6) (Abdel-Mottaleb et al., Toxicon 51:1424-30, 2008; Epub 2008 Mar. 29).
Engineered toxin peptides with improved potency, selectivity and/or half life including OsK1 and ShK have been reported (Int. Pat. Appl. Publ. WO2006/002850; Int. Pat. Appl. Publ. WO2006/042151; Int. Pat. Appl. Publ. WO2008/088422, Int. Pat. Appl. Publ. WO2006/116156).
There exists a need for more potent and selective Kv1.3 blockers for the therapeutic treatment of Kv1.3-mediated diseases such as T-cell mediated inflammatory and autoimmune diseases such as lupus and multiple sclerosis.